The present invention relates to the formation and methods for use of peptide conjugates which interact with specific, defined sites of biological activity in vivo or in vitro. More particularly, the invention relates to a method of forming biologically active molecules which react with a defined site in a vertebrate system and have an extended lifetime in the circulatory system.
The biochemical systems of a vertebrate body act by interaction of various biological molecules. Generally, the sites of interaction are relatively small even though the size of the total molecule involved in the interaction may be very large. For example, although many proteins such as enzymes have a molecular weight in the ten to one hundred thousand dalton range formed of hundreds to thousands of amino acid residues, the actual active site is normally comprised of one or a few specifically positioned amino acid residues. Because of the small size of these sites of biologic activity, small peptides which mimic the active site can be used to inhibit or act in place of the active sites of these larger molecules. These small peptides can react with a variety of protein or lipid molecules including binding proteins, inhibitory proteins, hormones, cofactors, activators and cell membrane sites (receptors). Because of this capacity to interact with functionally important sites, these peptides can be used as pharmaceutical agents.
The vertebrate body has a series of natural defenses, including the immune system, which clears the blood stream of a variety of molecules. A small peptide, for example twenty amino acid residues or less, is normally cleared within minutes. However, naturally occuring larger molecules, e.g., large proteins such as human serum albumin, immunoglobulins and other molecules which cause little or no immunogenic response in a vertebrate have an extended lifetime in the circulatory system; the lifetime may be from three to as much as forty-five days.
One of the problems with molecules which are cleared quickly from the bloodstream is that to obtain a pharmaceutical or other beneficial effect, a specific range of concentration is needed. To achieve this concentration range with molecules that are cleared quickly, two general methods of maintaining the level have been used; a mechanical device or a controlled release system. Mechanical devices such as infusion pumps or intravenous units are used to add material to the bloodstream at a rate which parallels the clearance rate of the material. The device obtains the material from a large reservoir and supplies it to the body at a constant rate.
The alternative is some form of controlled release implant or device. These controlled release devices are based on either of two distinct schemes: the material is trapped or bound in a device which has pores and allows the release of the material by an osmotic pressure method; or the material is trapped or bound in a material or membrane which is hydrolyzed or broken down slowly by chemical reactions in the bloodstream. Examples of the first of these methods are microcapsules or other forms of encapsulated hormone or drugs. Examples of the second type are such shown in Sidman patent, U.S. Pat. No. 4,351,337 which discloses a biodegradable implantable drug delivery system where enzymes destroy the walls of the carrier at a substantially controlled rate, allowing controlled rate release of the material into the bloodstream.
Both of these methods of attempting to achieve the substantially constant level of material in the bloodstream have attendant problems. The mechanical methods of achieving this end require a reservoir as well as possibly limiting the mobility of the infused animal or person. Mechanical methods also rely on devices which may break down in time. Methods for controlled rate release using implantable or oral receptacles which release the material based on osmotic pressure or by controlled hydrolysis or breakdown of a membrane also have substantial problems. These products usually do not have true zero order kinetic release and, therefore, there is some variation in the amount of material in the bloodstream of the animal. In osmotic devices, there is normally a higher outflow rate at the time of the implant while in the hydrolysis or breakdown methods, the initial rate will normally be lower while the final rate will be higher as hydrolysis or breakdown of the membrane releases more and more pockets of the material. Since most pharmaceutical preparations need an extended lifetime for activity, there would be an advantage in producing molecules which interact with the sites of biological activity, such as the small peptides, but have the extended lifetime of the larger, macromolecular molecules.
Accordingly, it is an object of the invention to provide a method of producing a biologically active material which has extended lifetime in the circulatory system without the need of mechanical infusion systems or degrading membranes. Another object of the invention is to provide a molecule which has specific activity with functionally important sites of proteins and lipids, particularly in cell membranes, while having an extended lifetime in the circulatory system. A further object of the invention is to provide a method of forming a synthetic molecule which will interact with defined sites of biological activity in a vertebrate and have an extended lifetime in the circulatory system. These and other objects and features of the inventions will be apparent from the summary of the invention, the drawing and the description.